Stx)-producing Escherichia Coli Strains Research Articles

Genetic Characterization of Intimin Gene (eae) in Clinical Shiga Toxin-Producing Escherichia coli Strains from Pediatric Patients in Finland.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections cause outbreaks of severe disease in children ranging from bloody diarrhea to hemolytic uremic syndrome (HUS). The adherent factor intimin, encoded by eae, can facilitate the colonization process of strains and is frequently associated with severe disease. The purpose of this study was to examine and analyze the prevalence and polymorphisms of eae in clinical STEC strains from pediatric patients under 17 years old with and without HUS, and to assess the pathogenic risk of different eae subtypes. We studied 240 STEC strains isolated from pediatric patients in Finland with whole genome sequencing. The gene eae was present in 209 (87.1%) strains, among which 49 (23.4%) were from patients with HUS, and 160 (76.6%) were from patients without HUS. O157:H7 (126, 60.3%) was the most predominant serotype among eae-positive STEC strains. Twenty-three different eae genotypes were identified, which were categorized into five eae subtypes, i.e., γ1, β3, ε1, θ and ζ3. The subtype eae-γ1 was significantly overrepresented in strains from patients aged 5-17 years, while β3 and ε1 were more commonly found in strains from patients under 5 years. All O157:H7 strains carried eae-γ1; among non-O157 strains, strains of each serotype harbored one eae subtype. No association was observed between the presence of eae/its subtypes and HUS. However, the combination of eae-γ1+stx2a was significantly associated with HUS. In conclusion, this study demonstrated a high occurrence and genetic variety of eae in clinical STEC from pediatric patients under 17 years old in Finland, and that eae is not essential for STEC-associated HUS. However, the combination of certain eae subtypes with stx subtypes, i.e., eae-γ1+stx2a, may be used as risk predictors for the development of severe disease in children.

Shiga Toxin (Stx) Phage-Encoded Lytic Genes Are Not Required for the Mouse Virulence of O157:H7 Escherichia coli Stx2-Producing Clinical Isolates.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne diarrheal illness in the United States and globally, and serotype O157:H7 is frequently associated with STEC outbreaks and sporadic cases in the United States. Severe systemic diseases associated with STEC are mediated by Stx types, particularly subtype Stx2a, encoded on inducible bacteriophages. We previously identified two STEC O157:H7 clinical isolates, JH2010 and JH2012, that exhibit a large difference in virulence in a streptomycin (Str)-treated mouse model. In this study, we aimed to identify a genetic basis for the difference in virulence between those strains. Comparison of the stx2a phage sequences showed that JH2012 lacks the lytic genes S and R on the phage genome. We also demonstrated that compared to JH2012 cultures, cultures of JH2010 released more Stx2 into the supernatant and were more sensitive to bacterial lysis during growth with ciprofloxacin (Cip), an inducer of stx phages. We therefore generated an stx2a phage SR deletion mutant strain of JH2010 to determine if those genes were responsible for the high virulence of that strain. We found that deletion of the SR genes from the stx2a phage in JH2010, and another O157:H7 strain, JH2016, resulted in increased cellular retention of Stx2, but there was no difference in virulence compared to the wild-type strains. Our results indicate that the stx2a phage SR genes are involved in Stx2 localization and phage-mediated cell lysis in vitro but that they are not required in wild-type STEC strains for virulence in a mouse model. IMPORTANCE The release of Stx from STEC has been thought to be tied to phage-mediated lysis of the host bacterial cell. In this study, we found that the stx2a phage lytic genes are not required for the virulence of pathogenic O157:H7 clinical isolates in a murine model of STEC infection or for release of Stx2a into the supernatant of bacterial cultures. These results point to an alternate mechanism for Stx2a release from STEC strains.

Shiga Toxin (Stx) Type 1a and Stx2a Translocate through a Three-Layer Intestinal Model.

Shiga toxins (Stxs) produced by ingested E. coli can induce hemolytic uremic syndrome after crossing the intact intestinal barrier, entering the bloodstream, and targeting endothelial cells in the kidney. The method(s) by which the toxins reach the bloodstream are not fully defined. Here, we used two polarized cell models to evaluate Stx translocation: (i) a single-layer primary colonic epithelial cell model and (ii) a three-cell-layer model with colonic epithelial cells, myofibroblasts, and colonic endothelial cells. We traced the movement of Stx types 1a and 2a across the barrier models by measuring the toxicity of apical and basolateral media on Vero cells. We found that Stx1a and Stx2a crossed both models in either direction. However, approximately 10-fold more Stx translocated in the three-layer model as compared to the single-layer model. Overall, the percentage of toxin that translocated was about 0.01% in the epithelial-cell-only model but up to 0.09% in the three-cell-layer model. In both models, approximately 3- to 4-fold more Stx2a translocated than Stx1a. Infection of the three-cell-layer model with Stx-producing Escherichia coli (STEC) strains showed that serotype O157:H7 STEC reduced barrier function in the model and that the damage was not dependent on the presence of the eae gene. Infection of the three-layer model with O26:H11 STEC strain TW08571 (Stx1a+ and Stx2a+), however, allowed translocation of modest amounts of Stx without reducing barrier function. Deletion of stx2a from TW08571 or the use of anti-Stx1 antibody prevented translocation of toxin. Our results suggest that single-cell models may underestimate the amount of Stx translocation and that the more biomimetic three-layer model is suited for Stx translocation inhibitor studies.

High Prevalence and Persistence of Escherichia coli Strains Producing Shiga Toxin Subtype 2k in Goat Herds.

ABSTRACTShiga toxin (Stx)-producing Escherichia coli (STEC) is a zoonotic pathogen with the ability to cause severe diseases like hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is the key virulence factor in STEC and can be classified into two types, Stx1 and Stx2, and different subtypes. Stx2k is a newly reported Stx2 subtype in E. coli strains from diarrheal patients, animals, and raw meats exclusively in China so far. To understand the reservoir of Stx2k-producing E. coli (Stx2k-STEC), we investigated Stx2k-STEC strains in goat herds and examined their genetic characteristics using whole-genome sequencing. A total of 448 STEC strains were recovered from 2,896 goat fecal samples, and 37.95% (170/448) were Stx2k-STEC. Stx2k-STEC strains of serotype O93:H28 and sequence type 4038 (ST4038) were the most predominant and were detected over several years. Notably, 55% of Stx2k-STEC strains carried the heat-labile toxin (LT)-encoding gene (elt) defining enterotoxigenic E. coli (ETEC), thereby exhibiting the hybrid STEC/ETEC pathotype. Stx2k-converting prophage genomes clustered into four groups and exhibited high similarity within each group. Strains from patients, raw meat, sheep, and goats were intermixed distributed in the phylogenetic tree, indicating the risk for cross-species spread of Stx2k-STEC and pathogenic potential for humans. Further studies are required to investigate the Stx2k-STEC strains in other reservoirs and to understand the mechanism of persistence in these hosts.IMPORTANCE Strains of the recently reported Stx2k-STEC have been circulating in a variety of sources over time in China. Here, we show a high prevalence of Stx2k-STEC in goat herds. More than half of the strains were of the hybrid STEC/ETEC pathotype. Stx2k-STEC strains of predominant serotypes have been widespread in the goat herds over several years. Stx2k-converting prophages have exhibited a high level of similarity across geographical regions and time and might be maintained and transmitted horizontally. Given that goat-derived Stx2k-STEC strains share similar genetic backbones with patient-derived strains, the high prevalence of Stx2k-STEC in goats suggests that there is a risk of cross-species spread and that these strains may pose pathogenetic potential to humans. Our study thus highlights the need to monitor human Stx2k-STEC infections in this region and, by extension, in other geographic locations.

The global population structure and evolutionary history of the acquisition of major virulence factor-encoding genetic elements in Shiga toxin-producing Escherichia coli O121:H19.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.

Comparative phenotypic characterization of hybrid Shiga toxin-producing / uropathogenic Escherichia coli, canonical uropathogenic and Shiga toxin-producing Escherichia coli

Hybrid Shiga toxin (Stx)-producing Escherichia coli (STEC) and uropathogenic E. coli (UPEC) strains are phylogenetically positioned between STEC and UPEC and can cause both diarrhea and urinary tract infections (UTIs). However, their virulence properties and adaptation to different host milieu in comparison to canonical UPEC and STEC strains are unknown.We determined phenotypes of the STEC/UPEC hybrid with respect to virulence including acid resistance, motility, biofilm formation, siderophore production, and adherence to human colonic Caco-2 and bladder T24 cells and compared to phenotypes of commensal strain MG1655, UPEC strain 536, and STEC strains B2F1 and Sakai. Moreover, we assessed the adaptation of the hybrid to artificial urine medium (AUM) and simulated colonic environment medium (SCEM).Overall acid resistance at pH 2.5 was high except in strains B2F1 and hybrid 05−00787 which showed reduced and extremely low acid resistance, respectively. Motility was reduced in hybrid 05−00787 and 09−05501 but strong in the remaining hybrids. While some hybrids showed high biofilm formation in LB, overall biofilm formation in SCEM and AUM were low and non-existent, respectively. All strains tested showed siderophore activity at equilibrium. All strains except MG1655 adhered to Caco-2 cells with the hybrid having similar adherence when compared to 536 but exhibited 2 and 3 times lower adherence when compared to B2F1 and Sakai, respectively. All Stx-producing strains adhered stronger to T24 cells than strains 536 and MG1655. Overall growth in LB, SCEM and AUM was consistent within the hybrid strains, except hybrid 05−00787 which showed significantly different growth patterns.Our data suggest that the hybrid is adapted to both, the intestinal and extraintestinal milieu. Expression of phenotypes typical of intestinal and extraintestinal pathogens thereby supports its potential to cause diarrhea and UTI.

Prevalence, O-genotype and Shiga toxin (Stx) 2 subtype of Stx-producing Escherichia coli strains isolated from Argentinean beef cattle.

The aims of this study were to investigate prevalence, O-genotype, and virulence gene profile including Shiga toxin (Stx) 2 gene-subtype of Stx-producing Escherichia coli (STEC) in beef cattle from the Bahía Blanca in Argentina. Rectal swabs were collected from 283 beef cattle in 2012. stx genes were detected in 90 (32%) out of the 283 rectal swabs by stx gene-specific PCR assay. The positive cases were 13 with stx1, 58 with stx2, and 19 with both stx1 and stx2. Among 90 stx gene-positive samples, 45 STEC strains were isolated, which included 3 stx1, 34 stx2, and eight stx1 and stx2 genes positive isolates. O-genotyping grouped 45 STEC strains into 19 different O-genotypes such as Og8, Og145, Og171, Og185 (4 from each), Og22, Og153, Og157 (3 from each) and others. Various stx2 gene-subtypes were identified in 42 STEC strains: 13 positive cases for stx2a, 11 for stx2c, 3 for stx2g, 10 for stx2a and stx2d, 4 for stx2a and stx2c, and 1 for stx2b, stx2c and stx2g. efaI gene, generally prevalent in clinical strains, was detected in relatively high in the STEC strains. These data suggest that stx2a and stx2c were distributed not only in O145 and O157 but also in minor O-genotypes of STEC in Argentina.

Enterotoxic, neurotoxic and cytotoxice demonstrated by shiga toxin (2d) producing escherichia coli in experimental models

Background: Shiga toxin (Stx) producing Escherichia coli (STEC) colonize human intestinal tract and their infections have asymptomatic clinical manifestations which cause local and systemic pathological changes.
 Objectives: This study intended to establish the role of Shiga toxin (Stx2d) in developing clinical manifestations in STEC infections using experimental models.
 Methods: A total 300 stool samples were screened from hospitalised diarrhoeal patients enrolled in 2% surveillance system at International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). The stx gene profile including their variants was identified byPCR.stx2d gene positive STEC PT187 was selected for toxin (s) preparation.Toxin was prepared by centrifugation of culture supernatant. Enterotoxicand paralytic-lethal activities were tested in rabbit ileal loops and mice, respectively. Histopathological study of the rabbit ileal loop segments and different tissues of mice by paraffin embedded method and stained by H & E staining. Cytotoxic effect was performed on HeLa cells.
 Results: Nine STEC strains were identified for stx2 gene positive. Among them STEC PT187 was found stx2d gene positive strain and selected for toxic activities. Toxin (s) responsible for causing accumulation of fluid in rabbit ileal loops and its segments showed inflammation and enterocytenecrosis. In mouse model, toxin(s) was found to cause hind limbs paralysis and death. Brain, spinal cord and kidney tissue of mice showed histopathological changes. Toxin (s) also showed positive cytotoxic activity in HeLa cell.
 Conclusion: In this study, results indicated that Stx2d producing E. coli exhibit not only enterotoxic activity, but also cause impaired neurological functions and cytotoxic effect.
 Bangladesh Med Res Counc Bull 2020; 46(1): 41-47

Antibody response to lipopolysaccharides and recombinant proteins of Shiga toxin (STX)-producing Escherichia coli (STEC) in children with haemolytic uraemic syndrome in Poland.

Typical haemolytic uraemic syndrome (STEC-HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), is a serious, life-threating disease that mainly affects children. Bacteriological and genetic tests are commonly used in the routine laboratory diagnosis of STEC-HUS; however, serological methods have emerged as useful and reliable diagnostic tools, especially when bacterial isolation fails. In this study, we present the results of the serological investigation of 72 paediatric patients suspected for HUS, hospitalized during 2011-2019 at the Department of Pediatrics and Nephrology of Children's Hospitals in Poland. During the routine laboratory investigation STEC strains were isolated only from nine stool samples. However, serological investigations confirmed 45 cases of STEC infections in children with HUS. In this study, 22 (48·9%) paediatric patients were infected by E. coli serotype O26, 11 (24·4%) by serotype O145, 9 (20·0%) by serotype O157, and 3 (6·7%) by E. coli serotype O111. In the majority of these patients, in addition to a high level of IgA, IgG and IgM antibodies to lipopolysaccharide of particular E. coli serotypes, antibodies to recombinant proteins Tir, Stx2b and intimin were detected. Our results confirm that serological tests are useful in the diagnosis of STEC-HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that serological analysis greatly complements bacterial isolation and helps in the diagnosis and confirmation of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) infections. Serological tests can be performed to qualify the patient for the typical haemolytic uraemic syndrome (STEC-HUS). In Poland, STEC-HUS in children is mostly caused by the E. coli serotype O26, which indicates that there is an increasing number of non-O157 STEC infections associated with human diseases in Europe.

Development and Evaluation of a Novel VHH-Based Immunocapture Assay for High-Sensitivity Detection of Shiga Toxin Type 2 (Stx2) in Stool Samples.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is the main cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening clinical complication characterized by hemolytic anemia, thrombocytopenia, and acute renal failure that mainly affects children. A relevant feature of STEC strains is the production of Stx, and all of them express Stx1 and/or Stx2 regardless of the strain serotype. Therefore, Stx detection assays are considered the most suitable methods for the early detection of STEC infections. Single-domain antibodies from camelids (VHHs) exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis. In this work, we have exploited VHH technology for the development of an immunocapture assay for Stx2 detection. Thirteen anti-Stx2 VHHs previously obtained from a variable-domain repertoire library were selected and evaluated in 130 capture-detection pair combinations for Stx detection. Based on this analysis, two VHHs were selected and a double VHH-based biotin-streptavidin capture enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection was developed and optimized for Stx2 detection. This assay showed an excellent analytical and clinical sensitivity in both STEC culture supernatants and stool samples even higher than the sensitivity of a commercial ELISA. Furthermore, based on the analysis of stool samples, the VHH-based ELISA showed high correlation with stx2 detection by PCR and a commercial rapid membrane-based immunoassay. The intrinsic properties of VHHs (high target affinity and specificity, stability, and ease of expression at high yields in recombinant bacteria) and their optimal performance for Stx detection make them attractive tools for the diagnosis of HUS related to STEC (STEC-HUS).

Molecular characteristics of eae-positive clinical Shiga toxin-producing Escherichia coli in Sweden

ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) can cause a wide range of symptoms from asymptomatic carriage, mild diarrhea to bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). Intimin, encoded by the eae gene, also plays a critical role in STEC pathogenesis. Herein, we investigated the prevalence and genetic diversity of eae among clinical STEC isolates from patients with diarrhea, BD, HUS as well as from asymptomatic STEC-positive individuals in Sweden with whole-genome sequencing. We found that 173 out of 239 (72.4%) of clinical STEC strains were eae positive. Six eae subtypes (ϵ1, γ1, β3, θ, ζ and ρ) were identified eae and its subtype γ1 were significantly overrepresented in O157:H7 strains isolated from BD and HUS patients. ϵ1 was associated with O121:H19 and O103:H2 strains, and β3 to O26:H11 strains. The combination of eae subtype γ1 and stx subtype (stx 2 or stx 1+stx 2) is more likely to cause severe disease, suggesting the possibility of using eae genotypes in risk assessment of STEC infection. In summary, this study demonstrated a high prevalence of eae in clinical STEC strains and considerable genetic diversity of eae in STEC strains in Sweden from 1994 through 2018, and revealed association between eae subtypes and disease severity.

Eleven High-Quality Reference Genome Sequences and 360 Draft Assemblies of Shiga Toxin-Producing Escherichia coli Isolates from Human, Food, Animal, and Environmental Sources in Canada.

We report high-quality closed reference genomes for 1 bovine strain and 10 human Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from serogroups O26, O45, O91, O103, O104, O111, O113, O121, O145, and O157. We also report draft assemblies, with standardized metadata, for 360 STEC strains isolated from watersheds, animals, farms, food, and human infections.

Combining Mass Spectrometry, Surface Acoustic Wave Interaction Analysis, and Cell Viability Assays for Characterization of Shiga Toxin Subtypes of Pathogenic Escherichia coli Bacteria.

Shiga toxin (Stx)-producing Escherichia coli (STEC) and enterohemorrhagic E.coli (EHEC) as a human pathogenic subgroup of STEC are characterized by releasing Stx AB5-toxin as the major virulence factor. Worldwide disseminated EHEC strains cause sporadic infections and outbreaks in the human population and swine pathogenic STEC strains represent greatly feared pathogens in pig breeding and fattening plants. Among the various Stx subtypes, Stx1a and Stx2a are of eminent clinical importance in human infections being associated with life-threatening hemorrhagic colitis and hemolytic uremic syndrome, whereas Stx2e subtype is associated with porcine edema disease with a generalized fatal outcome for the animals. Binding toward the glycosphingolipid globotriaosylceramide (Gb3Cer) is a common feature of all Stx subtypes analyzed so far. Here, we report on the development of a matched strategy combining (i) miniaturized one-step affinity purification of native Stx subtypes from culture supernatant of bacterial wild-type strains using Gb3-functionalized magnetic beads, (ii) structural analysis and identification of Stx holotoxins by electrospray ionization ion mobility mass spectrometry (ESI MS), (iii) functional Stx-receptor real-time interaction analysis employing the surface acoustic wave (SAW) technology, and (iv) Vero cell culture assays for determining Stx-caused cytotoxic effects. Structural investigations revealed diagnostic tryptic peptide ions for purified Stx1a, Stx2a, and Stx2e, respectively, and functional analysis resulted in characteristic binding kinetics of each Stx subtype. Cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e. Collectively, this matched procedure represents a promising clinical application for the characterization of life-endangering Stx subtypes at the protein level.

Pro-inflammatory capacity of Escherichia coli O104:H4 outbreak strain during colonization of intestinal epithelial cells from human and cattle

In 2011, Germany was struck by the largest outbreak of hemolytic uremic syndrome. The highly virulent E. coli O104:H4 outbreak strain LB226692 possesses a blended virulence profile combining genetic patterns of human adapted enteroaggregative E. coli (EAEC), rarely detected in animal hosts before, and enterohemorrhagic E. coli (EHEC), a subpopulation of Shiga toxin (Stx)-producing E. coli (STEC) basically adapted to the ruminant host. This study aimed at appraising the relative level of adaptation of the EAEC/EHEC hybrid strain LB226692 to humans and cattle. Adherence and invasion of the hybrid strain to intestinal (jejunal and colonic) epithelial cells (IEC) of human and bovine origin was compared to that of E. coli strains representative of different pathovars and commensal E. coli by means of light and electron microscopy and culture. Strain-specific host gene transcription profiles of selected cytokines and chemokines as well as host-induced transcription of bacterial virulence genes were assessed. The release of Stx upon host cell contact was quantified. The outbreak strain’s immunomodulation was assessed by cultivating primary bovine macrophages with conditioned supernatants from IEC infection studies with E. coli, serving as model for the innate immunity of the bovine gut. The outbreak strain adhered to IEC of both, human and bovine origin. Electron microscopy of infected cells revealed the strain’s particular affinity to human small IEC, in contrast to few interactions with bovine small IEC. The outbreak strain possessed a high-level of adhesive power, similar to human-associated E. coli strains and in contrast to bovine-associated STEC strains. The outbreak strain displayed a non-invasive phenotype, in contrast to some bovine-associated E. coli strains, which were invasive. The outbreak strain provoked some pro-inflammatory activity in human cells, but to a lower extent as compared to other pathotypes. In contrasts to bovine-associated E. coli strains, the outbreak strain induced marked pro-inflammatory activity when interacting with bovine host cells directly (IEC) and indirectly (macrophages). Among stx2-positive strains, the human-pathogenic strains (LB226692 and EHEC strain 86-24) released higher amounts of Stx compared to bovine-associated STEC. The findings imply that the outbreak strain is rather adapted to humans than to cattle. However, the outbreak strain’s potential to colonize IEC of both host species and the rather mixed reaction patterns observed for all strains under study indicate, that even STEC strains with an unusual genotype as the EHEC O104:H4 outbreak strain, i.e. with an EAEC genetic background, may be able to conquer other reservoir hosts.

The Gene tia, Harbored by the Subtilase-Encoding Pathogenicity Island, Is Involved in the Ability of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli Strains To Invade Monolayers of Epithelial Cells.

Locus of enterocyte effacement (LEE)-negative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are human pathogens that lack the LEE locus, a pathogenicity island (PAI) involved in the intimate adhesion of LEE-positive strains to the host gut epithelium. The mechanism used by LEE-negative STEC strains to colonize the host intestinal mucosa is still not clear. The cell invasion determinant tia, previously described in enterotoxigenic E. coli strains, has been identified in LEE-negative STEC strains that possess the subtilase-encoding pathogenicity island (SE-PAI). We evaluated the role of the gene tia, present in these LEE-negative STEC strains, in the invasion of monolayers of cultured cells. We observed that these strains were able to invade Caco-2 and HEp-2 cell monolayers and compared their invasion ability with that of a mutant strain in which the gene tia had been inactivated. Mutation of the gene tia resulted in a strong reduction of the invasive phenotype, and complementation of the tia mutation with a functional copy of the gene restored the invasion activity. Moreover, we show that the gene tia is overexpressed in bacteria actively invading cell monolayers, demonstrating that tia is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli, including STEC, strains. However, the expression of the tia gene in the E. coli K-12 strain JM109 was not sufficient, in its own right, to confer to this strain the ability to invade cell monolayers, suggesting that at least another factor must be involved in the invasion ability displayed by the SE-PAI-positive strains.

Ouabain Protects Human Renal Cells against the Cytotoxic Effects of Shiga Toxin Type 2 and Subtilase Cytotoxin

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2) are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB) is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA) may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC) and the human proximal tubule epithelial cell (HK-2) line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS.

Distinct Expression of Immunoglobulin-Binding Proteins in Shiga Toxin-Producing Escherichia coli Implicates High Protein Stability and a Characteristic Phenotype.

Several immunoglobulin-binding proteins of Escherichia coli (Eib) have been isolated from both non-pathogenic and pathogenic E. coli strains. Shiga toxin (Stx)-producing E. coli (STEC) contain eibG either as a single gene or in combination with eibC, while other E. coli strains harbour single or multiple eib genes. The Eib proteins bind human immunoglobulins in a non-immune manner and contribute to bacterial chain-like adherence to human epithelial cells. In this study, the EibG expression in several STEC strains was analysed under different environmental conditions. STEC produced high levels of EibG in complex media and lower levels in low-grade and minimal media under static growth conditions. This characteristic was independent on the Eib subtypes. Microscopically, EibG-expressing STEC exhibited chain formation and aggregation in all employed media, while aggregates were only visible after growth in complex medium. Once expressed, EibG proteins demonstrate high stability during prolonged incubation. Our findings indicate that the regulation of the expression of Eib proteins is highly complex, although the protein levels vary among STEC strains. However, positive upregulation conditions generally result in distinct phenotypes of the isolates.

Genetic makeup of Shiga toxin-producing Escherichia coli in relation to clinical symptoms and duration of shedding: a microarray analysis of isolates from Swedish children

Shiga toxin (Stx)-producing Escherichia coli (STECs) cause non-bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome, and are the primary cause of acute renal failure in children worldwide. This study investigated the correlation of genetic makeup of STEC strains as revealed by DNA microarray to clinical symptoms and the duration of STEC shedding. All STEC isolated (n = 96) from patients <10 years of age in Jönköping County, Sweden from 2003 to 2015 were included. Isolates were characterized by DNA microarray, including almost 280 genes. Clinical data were collected through a questionnaire and by reviewing medical records. Of the 96 virulence genes (including stx) in the microarray, 62 genes were present in at least one isolate. Statistically significant differences in prevalence were observed for 21 genes when comparing patients with bloody diarrhea (BD) and with non-bloody stool (18 of 21 associated with BD). Most genes encode toxins (e.g., stx2 alleles, astA, toxB), adhesion factors (i.e. espB_O157, tir, eae), or secretion factors (e.g., espA, espF, espJ, etpD, nleA, nleB, nleC, tccP). Seven genes were associated with prolonged stx shedding; the presence of three genes (lpfA, senB, and stx1) and the absence of four genes (espB_O157, espF, astA, and intI1). We found STEC genes that might predict severe disease outcome already at diagnosis. This can be used to develop diagnostic tools for risk assessment of disease outcome. Furthermore, genes associated with the duration of stx shedding were detected, enabling a possible better prediction of length of STEC carriage after infection.

Expression of Shiga toxin 2 (Stx2) in highly virulent Stx-producing Escherichia coli (STEC) carrying different anti-terminator (q) genes

Shiga toxins (Stx) are key virulence factors of Shiga toxin-producing Escherichia coli (STEC) during development of haemolytic uremic syndrome (HUS). It has been suggested that not only specific stx2 subtypes, but also the amount of Stx2 expressed might be essential for STEC pathogenicity. We aimed to investigate if various anti-terminator (q) genes might influence the expression level of Stx2 in highly virulent STEC.A multiplex PCR detecting q933, q21, and qO111 was run on 20 stx2a-positive STEC strains, of which 18 were HUS associated serotypes (HAS) and two non-HAS. Relative expression of Stx2 mRNA was assessed for all strains, both in non-induced and induced (mitomycin C) state.The HAS STEC carried either q933 (n = 8), qO111 (n = 8), or both (n = 2). In basal state, no STEC strains showed higher expression of Stx2 mRNA than the calibrator EDL933 (non-sorbitol fermenting (NSF) O157:H7carrying q933). Variations among strains were not associated with different q genes present, but rather related to specific serogroups. In induced state, O104:H4 strains (q933) showed higher Stx2 mRNA level than EDL933, whereas sorbitol fermenting (SF) O157:H- (qO111) and O121:H? (q933) STEC showed levels comparable with EDL933. An association between the presence of q933 and higher Stx2 level was seen within some HAS, but not all. Interestingly, the O103:H25 STEC strains, responsible for a HUS outbreak in Norway, carried both q933 and qO111. However, the Stx2 mRNA level in these strains was significantly lower than EDL933 in both states, indicating that other factors than the level of Stx2 might explain the aggressiveness of these bacteria. The two non-HAS STEC did not carry any of the examined q genes. In induced state, these bacteria showed the lowest Stx2 mRNA level compared to EDL933. One of the non-HAS STEC was not induced by mitomycin C, suggesting that stx2a might be located on a defect bacteriophage.No association between specific q genes and Stx2 mRNA expression level was revealed in stx2a-positive HAS STEC. Our results suggest that other factor(s) than specific q genes might influence the level of Stx2 produced in highly virulent STEC.

Virulence Potential of Activatable Shiga Toxin 2d-Producing Escherichia coli Isolates from Fresh Produce

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named “activation.” Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin- treated mice.